Bacteriophages

Bacteriophages
DR. KAVEH HARATIAN
MASTER EDUCATION SERIES - AUMS - 2014 1

What do you think about them
?

Some interesting facts
Viruses that infect bacteria.
Viruses of dsDNA(tailed dsDNA phages).easily in environmental samples
107 /mL in coastal sea-water
1031 individual tailed phage virion on planet Earth.
End to end : 200 million light years into intergalactic space
Most bacterial genomes contain 1 to 24 phage genome as prophage
Turn over every 4-5 years: 1024 productive infections/sec

What are Bacteriophages ?
Viruses that attack bacteria were observed by
Twort and d'Herelle in 1915 and 1917. They
observed that broth cultures of certain intestinal
bacteria could be dissolved by addition of a
bacteria-free filtrate obtained from sewage

Brief history
Discovered in 1915/1917. basis on :
◦ Clearing(cell lysis) in bacterial lawn.
◦ Propagation as infectious agents
Ideas:
◦ Nature of viruses
◦ Phage therapy: using phages as an agent to combat bacterial infections
Modern era of phage research, 1940:
◦ Nature of gene
◦ Using phages as experimental model system
Extra results:
◦ Nature of gene
◦ Expression of genes and expression regulation
◦ Development of methods of recombinant DNA.

Bacteriophages under Electron
Microscope

Bacteriophage (Phage)
Definition - Obligate intracellular parasites that
multiply inside bacteria by making use of some or
all of the host biosynthetic machinery
Significance
◦ Models for animal cell viruses
◦ Gene transfer in bacteria
◦ Medical applications
◦ Identification of bacteria - phage typing
◦ Treatment and prophylaxsis???

Bacteriophages as therapeutic
agents
1919: successful treatment of typhoid in chickens , dysentery in humans.
1921: using against staphylococcus in skin disease.
1920s: large-scale experiments in many countries, like india.
1939 upward:
◦ Against diseases with no bacterial component: herpes, urticaria
◦ Although high specific, were used against inappropriate bacterial targets
◦ Inappropriate growth condition or preservatives could limit/prevented the inclusion of
infectious Bacteriophages.
◦ No good evidence that they actually worked in the therapeutic uses.
◦ But it continued through WW2(German and soviet armies) notably against dysentery.
Antibiotic age
Renewal of interest

Bacteriophages:
Definition & History
Bacteriophages are viruses that can
infect and destroy bacteria.
They have been referred to as bacterial
parasites, with each phage type
depending on a single strain of bacteria
to act as host.

BACTRIOPHAGES
Like most viruses, bacteriophages typically
carry only the genetic information needed
for replication of their nucleic acid and
synthesis of their protein coats.. They
require precursors, energy generation and
ribosomes supplied by their bacterial host
cell.

Bacteriophages:
Classification
At present, over 5000
bacteriophages have
been studied by
electron microscopy
and can be divided
into 13 virus families.

ICTV-designated phage families
Family Prototypes Characteristics
Siphoviridae λ dsDNA, long, noncontractile tails
Myoviridae T4 dsDNA, contractile tails
Podoviridae T7 dsDNA, short, stubby tails
Tectiviridae PRD1 dsDNA, linear 5` proteins, internal membrane
Microviridae φX174 ssDNA, circular, icosahedral
Inoviridae M13 ssDNA, filamentous
Leviviridae MS2, Qβ ssDNA, small icosahedral
Cystoviridae φ6 dsDNA, segmented, enveloped
Corticoviridae PM2 dsDNA, circular, internal membrane
Plasmaviridae L2 dsDNA, circular, enveloped

Bacteriophage
Bacteriophages
make up a diverse
group of viruses,
some of which have
complex structures,
including double-
stranded DNA.

Bacteriophage

Bacteriophage
Also known simply as a
phage; a virus that attacks
and infects bacteria. The
infection may or may not lead
to the death of the
bacterium, depending on the
phage and sometimes on
conditions. Each
bacteriophage is specific to
one form of bacteria.

Composition and Structure
Composition
◦Nucleic acid
◦Genome size
◦Modified bases
◦Protein
◦Protection
◦Infection
• Structure (T4)
– Size
– Head or capsid
– Tail
Tail
Tail Fibers
Base Plate
Head/Capsid
Contractile
Sheath

Phage entering a bacterial cell

Virulent vs. temperate phages
• Virulent phages do not integrate their genetic
material into the host cell chromosome and
usually kill the host cells (lytic infection)
(e.g. T-phages of E.coli).
• Temperate phages may integrate into the host
DNA, causing LYSOGENY.

Bacteriophage showing Lytic and
lysogenic cycle

Bacteriophages:
Virulence Factors Carried On Phage
Temperate phage can go through one of two life
cycles upon entering a host cell.
1) Lytic:
Is when growth results in lysis of the host and release of
progeny phage.
2) Lysogenic:
Is when growth results in integration of the phage DNA into
the host chromosome or stable replication as a plasmid.
Most of the gene products of the lysogenic phage remains
dormant until it is induced to enter the lytic cycle.

Bacteriophages:
Lysogenic Conversion
Some lysogenic phage carry genes that can
enhance the virulence of the bacterial host.
 For example, some phage carry genes that encode toxins.
These genes, once integrated into the
bacterial chromosome, can cause the once
harmless bacteria to release potent toxins
that can cause disease.

Bacteriophages
• Used for cloning foreign
genes among other
applications
• Proteins and peptides are
fused to the
Capsid(surface) of the
phage
• The combination of the
phage and peptide is
known as a Fusion Protein

Lytic and Lysogenic cycle

Bacteriophages:
Lysogenic Conversion
Bacterium Phage
Gene
Product
Phenotype
Vibrio cholerae CTX phage cholerae toxin cholera
Escherichia coli
lambda
phage
shigalike toxin
hemorrhagic
diarrhea
Clostridium botulinum
clostridial
phages
botulinum
toxin
botulism (food
poisoning)
Corynebacterium
diphtheriae
corynephage
beta
diphtheria
toxin
diphtheria
Streptococcus
pyogenes
T12
erythrogenic
toxins
scarlet fever
Examples of Virulence Factors Carried by Phage

Lysogenic conversion
In some interactions between lysogenic phages
and bacteria, lysogenic conversion may occur. It
is when a temperate phage induces a change in
the phenotype of the bacteria infected that is
not part of a usual phage cycle. Changes can
often involve the external membrane of the cell
by making it impervious to other phages or even
by increasing the pathogenic capability of the
bacteria for a host.

Assay for Lytic Phage
• Plaque assay
– Method
– Plaque forming unit
(pfu)
– Measures infectious
particles
Bacteria
Phage
+
Phage
Plaque assay
Method
Plaque forming
unit (pfu)
Measures
infectious particles

Lytic
vs
Lysogenic
Cycle

Transduction

Figure 13.10.1
Attachment:
Phage
attaches to
host cell.
Penetration:
Phage pnetrates
host cell and
injects its DNA.
Synthesis of viral
compartments
1
2
3
Bacterial
cell wall
Bacterial
chromosome
Capsid DNA
Capsid
Sheath
Tail fiber
Base plate
Pin
Cell wall
Tail
Plasma membrane
Sheath contracted
Tail core

Figure 13.10.2
4 Maturation:
Viral components
are assembled
into virions.
Tail
5 Release:
Host cell lyses
and new virions
are released.
DNA
Capsid
Tail fibers

Examples:
* Corynebacterium diphtheria produces the toxin of diphtheria only when it
is infected by the phage β. In this case, the gene that codes for the toxin is
carried by the phage, not the bacteria.
* Vibrio cholerae is a non-toxic strain that can become toxic, producing
cholera toxin, when it is infected with the phage CTXφ.
* Clostridium botulinum causes botulism.
* Streptococcus pyogenes causes scarlet fever.
* Shiga toxin
* Tetanus

Medical Applications of Phages
“I strongly believe phage could become an
effective antibacterial tool” - Carl Merril, Chief of
the Laboratory of Biochemical Genetics, National Institute
of Mental Health, NIH.
“It might be another string on the bow, such
that when (conventional antibiotics) fail,
here’s something that has a chance of
working. But it’s not going to be a panacea” -
Joshua Lederberg, Sackler Foundation Scholar at The
Rockefeller University

Model Organisms

Model Organisms
Fundamental problems are solved in
the simplest and most accessible system
in which the problem can be addressed.
These organisms are called model
organisms.

Some Important Model
Organisms
Escherichia coli and its phage (the T phage and phage λ)
Baker’s yeast Saccharomyces cerevisiae
The nematode Caenorhabditis elegans
The fruit fly Drosophila melanogaster
The house mouse Mus musculus

Features of Model Systems
The availability of powerful tools of
traditional and molecular genetics.
The study of each model system
attracted a critical mass of investigators.
(Ideas,methods, tools and strains could
be shared)

HOW to choose a model
organism?
It depends on what question is being asked.
When studying fundamental issues of
molecular biology, simpler unicellular
organisms or viruses are convenient. For
developmental questions, more complicated
organisms should be used.

Model 1:
BACTERIOPHAGE

Bacteriophage (Viruses)
The simplest system
Their genomes are replicated only
after being injected into a host cell.
The genomes can recombine during
these infections.

Figure BacteriophageMASTER EDUCATION SERIES - AUMS - 2014 46

Each phage attaches to a specific cell
surface molecule (usually a protein)
and so only cells bearing that
“receptor” can be infected by a given
phage.

Two Basic Types
1. Lytic phage: eg. T phage
infect a bacterial cell
DNA replication
coat proteins expression
host cell lysed to release the new
phage

Figure 21-1
The lytic growth cycle

2. Temperate phage:
eg. Phage λ
Lysogeny—the phage genome integrated into
the bacterial genome and replicated passively
as part of the host chromosome, coat protein
genes not expressed.
•The phage is called a prophage.
•Daughter cells are lysogens.

Figure 21-2
The lysogenic
cycle of a
bacteriophage

The lysogenic state can switch to lytic
growth, called induction.
Excision of the prophage DNA
DNA replication
Coat proteins expression
Lytic growth

Figure 16-24
Growth and
induction of λ
lysogen

Assays of Phage Growth
Progagate phage:
by growth on a suitable
bacterial host in liquid
culture.
Quantify phage:
plaque assay
Bacteriophage

Progagate phage
Find a suitable
host cell that
supports the
growth of the virus.
The mixture of
viruses and
bacteria are filtered
through a bacterial-
proof filter.

Quantify phage
Phage are mixed with and adsorb to
bacterial cells.
Dilute the mix.
Add dilutions to “soft agar” (contain many
uninfected bacterial cells).
Poured onto a hard agar base.
Incubated to allow bacterial growth and
phage infection.

Soft
agar
Hard agar
a petri dish

This circle-of-death produces a hole or
PLAQUE in a lawn of living cells. These
plaques can be easily seen and counted so
that the numbers of virus can be quantitated.
As the viruses
replicate and
are released,
they spread
and infect the
nearby cells.

The Single-Step Growth Curve
Bacteriophage
Figure 21-4
Latent period-
the time lapse
between
infection and
release of
progeny.
Burst size-the
number of
phage released

The Single-Step Growth Curve
It reveals the life cycle of a typical lytic
phage.
It reveals the length of time it takes a
phage to undergo one round of lytic growth,
and also the number of progeny phage
produced per infected cell.

Method
1. Phage were mixed with bacterial cells for
10 minutes. (Long enough for adsorption
but too short for further infection
progress.)
2. The mixture is diluted by 10,000. (Only
those cells that bound phage in the initial
incubation will contribute to the infected
population; progeny phage produced from
those infections will not find host cells to
infect.)

3. Incubate the dilution. At intervals, a sample can
be removed from the mixture and the number of
free phage counted using a plaque assay.

Phage Crosses and
Complementation Tests
Bacteriophage
Mixed infection: a single
cell is infected with two
phage particles at once.

Mixed infection (co-infection)
1. It allows one to perform phage crosses.
If two different mutants of the
same phage co-infect a cell,
recombination can occur between
the genomes. The frequency of
this genetic exchange can be used
to order genes on the genome.

2. It allows one to assign
mutations to complementation
groups.
If two different mutant phage co-infect
the same cell and as a result each
provides the function that the other
was lacking, the two mutations must be
in different genes (complementation
groups). If not, the two mutations are
likely located in the same gene.

Transduction and
Recombinant DNA
Bacteriophage
During infection, a phage might pick
up a piece of bacterial DNA (mostly
happens when a prophage excises form
the bacterial chromosome).
The resulting recombinant phage can
transfer the bacterial DNA from one
host to another, known as specialized
transduction.
eg. Phage λMASTER EDUCATION SERIES - AUMS - 2014 66

This series Created by Dr.Kaveh Haratian Ph.D. for Medical and Master
learning.
Email
astrokaveh@gmail.com

Bacteriophages

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